Abstract

RATIONALE:

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator gene, which codes for a chloride channel, but the role of this chloride channel in inflammation induced by lung infection with Pseudomonas aeruginosa remains to be defined.

OBJECTIVES:

We tested the hypothesis that loss of this chloride channel alone is sufficient to cause excessive inflammation in response to inflammatory stimuli.

METHODS:

We investigated the response of cystic fibrosis and wild-type mice to mucoid P. aeruginosa administered by insufflation.

MEASUREMENTS:

MAIN RESULTS:

Depending on the dose administered and frequency of dosing, cystic fibrosis mice experienced significantly higher mortality rates, greater weight loss, higher lung pathology scores, and higher inflammatory mediator and neutrophil levels compared with wild-type mice, even after the bacteria had been cleared. Surprisingly, bacteria were cleared just as rapidly in cystic fibrosis mice as in wild-type mice, and sepsis was not observed. Chronic lung infections could not be established with mucoid P. aeruginosa in either cystic fibrosis or wild-type mice.

CONCLUSIONS:

Absence of this chloride channel alone appears sufficient for exaggerated inflammation and excess mortality compared with wild-type controls in the face of mucoid P. aeruginosa lung infection. To establish chronic infection, additional factors such as bacterial trapping or poor clearance may be required.

Weight change after a single inoculation with P. aeruginosa. Cystic fibrosis (CF; open symbols) and wild-type (closed symbols) mice were inoculated on Day 0 with P. aeruginosa by insufflation (indicated by the arrow). (A) Data are from a single experiment. Mice received approximately 108 cfu (6 mice/group), were weighed daily, and survivors were killed on Day 6. (B) Data were combined from six experiments. Mice were killed at each time point, leading to the decline in the number of mice over time. Mice received approximately 5 × 107 cfu, and weighed 1 (71 mice/group), 2 (53 mice/group), 3 (23–26 mice/group), and 4 d (12–13/group) after infection. Three CF mice died in the group to be killed on Day 3, and one died in the group to be killed on Day 4. *Significantly different (p ⩽ 0.006) from wild-type mice using a two-way analysis of variance (ANOVA) model to test for differences between the groups while controlling for the experiment. The Bonferroni method was used to adjust for multiple testing; a p value < 0.01 was considered significant. (C) Wild-type and (D) CF mice (n = 6/group) were inoculated with either live bacteria (circles), gluteraldehyde-fixed bacteria (triangles), or heat-killed bacteria (squares). Reductions in sample sizes indicated in A and D are due to spontaneous death due to overwhelming pulmonary infection. As a result of censorship by death, statistical analysis was not performed.

Leukocytes in bronchoalveolar lavage fluid (BALF) after a single inoculation with P. aeruginosa. Wild-type (closed circles) and CF mice (open triangles) were inoculated with a single dose of P. aeruginosa. Mice were killed 3 h, (n = 18/group), 1 d (18 mice/group), 2 d (27 mice/group), 3 d (11–13 mice/group), and 4 d (12–13/group) after infection (B), and represent mice from Figure 1B. BAL was performed and relative or differential (A, C, and E) and absolute or total numbers (B, D, and F) of alveolar macrophages (A and B), neutrophils (C and D), and lymphocytes (E and F) were enumerated, as described. *Significantly different (p ⩽ 0.009) from wild-type control mice at the same time point (due to data that were not normally distributed, the Van Elteren's test was used adjusting for differences between experiments and the Bonferroni method was used to adjust for multiple testing; p < 0.01 was considered significant). Arrows indicate the timing of infection. Data points at t = 0 represent untreated mice.

Bacterial numbers in lung homogenates after a single inoculation with P. aeruginosa. CF (open bar, open triangles) and wild-type (WT) mice (solid bar, closed circles) were inoculated with a single dose of P. aeruginosa. (A) WT and CF mice (n = 26 and 27, respectively; data combined from four experiments) were killed 3 h after infection (average, 6 × 107 cfu/mouse). (B) Mice were killed immediately, 1 h, or 2 h after infection (average, 4 × 107 cfu/mouse, n = 12/group). Data are combined from two experiments. There were no significant differences between the CF and WT mice at any time point studied (Van Elteren's test was used adjusting for differences between experiments).

Change in body weight after repeated administrations of P. aeruginosa. CF and wild-type mice were inoculated with P. aeruginosa by insufflation (arrows), and weighed daily. (A) CF (open triangles) and wild-type mice (closed circles) were inoculated on Days 0, 3, 6, and 9 (∼ 5 × 107 cfu/mouse; n = 10–11/group), and killed on Day 13. All but one CF mouse survived to the termination of the study. *Significantly different differences in change in body weight from wild-type mice (p ⩽ 0.038; unpaired Student's t test, the Bonferroni method was used to adjust for multiple testing). (B) CF (open triangles) and wild-type mice (closed circles) were inoculated on Days 0 (5 × 107 cfu/mouse), 2 (108 cfu/mouse), and 4 (3 × 107 cfu/mouse; n = 15/group), and killed on Day 8. Changes in sample size indicated in the figure are due to death. *Significant differences in change in body weight from wild type mice comparing groups where data are not censored by death (p = 0.009, unpaired Student t test; the Bonferroni method was used to adjust for multiple testing on Days 1 and 2).

Dose response study. CF mice were inoculated on Days 0, 2, and 4 with a low (∼ 5 × 105 cfu/mouse; closed circles or open bars), medium (∼ 5 × 106 cfu/mouse; open triangles or hatched bars), or high (∼ 5 × 107 cfu/mouse; closed squares or cross-hatched bars) dose of P. aeruginosa, weighed daily and killed on Day 7 (n = 10/group). All mice survived to the termination of the study. After the mice were killed, BAL was performed for cytokine analysis and cell counts. (A) Change in body weight after infection. (B) KC concentration in ELF. (C) Relative cell numbers of alveolar macrophages (AM), neutrophils (PMN), and lymphocytes (Lymph) in BALF. (D) Absolute cell counts of leukocytes (Leuk), AM, PMN, and Lymph in BALF. *Significantly different (p < 0.001 using a one way ANOVA, and p < 0.001 using a Tukey test to make pairwise comparisons) from the other two groups. †Significantly different from mice receiving the other two dosing groups (p < 0.001 using the Kruskal-Wallis test and p < 0.05 using Dunn's method to make pairwise comparisons). The Bonferroni method was used to adjust for multiple comparisons.

Inflammation after repeated administrations of P. aeruginosa. CF (open bars) and wild-type (WT) mice (closed bars) were inoculated with P. aeruginosa by insufflation (∼ 5 × 107 cfu/mouse) on Days 0 and 2, and killed on Day 4 (n = 15/group). All but one CF mouse survived the duration of the experiment, and BALF was not processed for cytokines from one CF mouse due to technical difficulties. (A) Change from initial body weight. (B) Inflammatory mediator concentration in ELF (n = 13–15/group). (C) Relative and (D) absolute cell counts in BALF (n = 14–15/group). (E) The area of inflamed lung was evaluated in seven mice from each group. *Significantly different from CF mice (p < 0.05 was considered significant; unpaired Student's t test or Mann-Whitney rank sum test).

Lung histology sections after repeated administrations of P. aeruginosa. Wild-type (A and E) and CF (B and F) mice were inoculated intranasally with P. aeruginosa on Day 0 (2.6 × 107 cfu) and Day 2 (6.9 × 108 cfu), and killed on Day 4. Lung sections from untreated wild-type (C and G) and CF (D and H) mice in similar areas of the lung are shown for comparison. BAL was performed and the lungs were prepared for sectioning and stained with alcian blue and periodic acid–Schiff (PAS). A magenta color indicates the presence of neutral polysaccharides (PAS positive), whereas acid mucopolysaccharides and 1,2-glycol acid substrates stain a dark blue (alcian blue positive; arrows). Endobronchial inflammation can be seen in the lumen of airway from a wild-type and CF mouse (A and B). Original magnification, 20× (A–D) and 40× (E–H); bar = 100 μm.